1. Field of the Invention
The present invention relates to a gel electrophoretic apparatus for separating and analyzing a biopolymer such as protein or nucleic acid, and more particularly, it relates to a capillary electrophoretic apparatus employing a capillary tube.
Such a capillary electrophoretic apparatus is employed in the biochemical field and for clinical testing, in particular as a DNA sequencer to obtain excellent results.
2. Description of the Prior Art
A DNA sequencer having high sensitivity, high speed and high throughput is necessary for sequence determination for DNA such as a human genome having long base sequence. For example, capillary electrophoresis employing a capillary column charged with a polymer serving as a separation medium is performed in place of slab gel electrophoresis employing a flat plate type slab gel. The separation medium is formed by a polymer and may be referred to as a polymer. With such a capillary column, a sample can not only be readily handled or injected, but also electrophoresed at a high speed to be detected in high sensitivity as compared to the slab gel. If a high voltage is applied to the slab gel, a band is spread or a temperature gradient is caused due to influence by Joulean heat. However, the capillary column hardly causes such a problem but can perform detection in high sensitivity with small band spreading even if performing high-speed electrophoresis with application of a high voltage.
A multi-capillary DNA sequencer prepared by arranging a plurality of capillary columns is also proposed.
In the capillary electrophoresis, a capillary column is charged with a polymer in the exterior of a capillary electrophoretic apparatus and thereafter mounted on the capillary electrophoretic apparatus. The capillary column and the polymer once used are discarded after electrophoresis and analysis. Thus, the running cost is increased, and the capillary column charged with a polymer having low viscosity is difficult to operate.
In order to reduce a cost and simplify handling of a capillary column charged with a polymer, there are proposed the following apparatuses: an apparatus (prior art 1) employing a polymer based on a cross-liking gel, and comprising a mechanism for reuse the polymer, a capillary column reuse type apparatus (prior art 2) comprising a mechanism charging and exchanging a polymer through a gas pressure, and a capillary column reuse type apparatus (prior art 3) comprising a syringe charge type mechanism for switching a valve mechanism and bringing a polymer into contact with a buffer solution, and the like.
In the prior art 1, it is difficult to charge the capillary column with the cross-linking gel with high yield since the inner part of the capillary column must be coated and bubbles may be mixed when reusing the cross-linking gel.
The prior art 2 requiring high-pressure gas of about 70 kgf/cm2 is difficult to operate. In particular, a multi-capillary lectrophoretic apparatus simultaneously handling a plurality of capillary columns requires a higher pressure and is more difficult to operate.
In the prior art 3, the polymer may leak from the valve mechanism when charged by a syringe with a high liquid pressure, to reduce the yield. In a multi-capillary electrophoretic apparatus, the mechanism is disadvantageously complicated.
Accordingly, an object of the present invention is to provide a capillary electrophoretic apparatus comprising a polymer charging mechanism capable of readily charging a capillary column with a polymer in a high yield.
The capillary electrophoretic apparatus according to the present invention comprises a capillary electrophoretic part electrophoresing a sample injected into a capillary column charged with a polymer, a detection part detecting each component separated in the capillary column on an appropriate position of the capillary column, a polymer syringe charged with a polymer, a pump block connecting the polymer syringe with one end of the capillary column through a passage, and a high voltage power supply device having electrodes provided on the one end and the other end of the capillary column so that the electrode provided on the one end comes into contact with the polymer for applying an electrophoresis voltage between the both ends of the capillary column with the electrodes.
In one aspect of the present invention, the pump block comprises a polymer port connected with the polymer syringe, a column port connected with the one end of the capillary column and a passage connecting the polymer port and the column port with each other, and the electrode on the one end of the capillary column is provided to come into contact with the polymer in the polymer syringe or the pump block.
In the capillary electrophoretic apparatus according to this aspect, the one end of the capillary column and the polymer syringe are connected to the pump block, and a piston of the polymer syringe is thereafter pushed to charge the capillary column with the polymer charged in the syringe through the passage of the pump block. Thereafter, a voltage is applied between both ends of the capillary column through the electrodes provided on both ends of the capillary column while connecting the capillary column with the pump block.
In another aspect of the present invention, the capillary electrophoretic apparatus further comprises a buffer syringe charged with a buffer, the pump block comprises a polymer port connected with the polymer syringe, a buffer port connected with the buffer syringe, a column port connected with the one end of the capillary column and a passage connecting the ports with each other, and the electrode on the one end of the capillary column is provided to come into contact with the polymer through the buffer.
It is preferable to form an openable drain port on the pump block and connect this drain port to the passage of the pump block.
In the capillary electrophoretic apparatus according to this aspect, after the drain port is opened, a piston of the buffer syringe is pushed while a piston of the polymer syringe is fixed, thereby air in the passage between the buffer syringe and the drain port is discharged through the drain port and the passage is charged with the buffer. Then, the piston of the buffer syringe is fixed and the piston of the polymer syringe is pushed while the drain port is kept open, thereby the buffer in the passage between the polymer syringe and the drain port is discharged through the drain port, and the passage is charged with the polymer. After the drain port is sealed, the piston of the polymer syringe is pushed and the piston of the buffer syringe is pulled in response to the amount of pushing, thereby the passage between the polymer syringe and the buffer syringe is charged with the polymer. Thereafter, the piston of the polymer syringe is pushed while the buffer syringe is fixed, thereby the passage between the polymer syringe and the capillary column as well as the capillary column are charged with the polymer.
It is preferable to comprise a pressure sensor measuring the pressure in the passage of the pump block. It is possible to charge the polymer, while monitoring the pressure in the passage with the pressure sensor for previously detecting abnormality such as clogging of the capillary column from the pressure value.
It is preferable to further comprise a tray storing a sample, a buffer and purified water, and a tray driving mechanism moving the tray for inserting the other end of the capillary column and the electrode into the sample, the buffer or the purified water. Consequently, sample injection, and separation and analysis after polymer charging can be automated by controlling the applied voltage and operations of the tray driving mechanism.
Thus, according to the present invention, the polymer syringe and the capillary column are connected by the passage in the pump block, and the syringe is pushed to charge the capillary column with the polymer, whereby the capillary column can be readily charged with the polymer in a high yield.
The foregoing and other objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of the present invention when taken in conjunction with the accompanying drawings.